Mini protean tgx 7.5 gels

For tissues, frozen skin and esophagus samples were minced into small pieces on ice using razor blades, homogenized in 300 μL RIPA buffer (25mM Tris pH 8, 150mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100) with protease and phosphatase inhibitors using a plastic pestle (Axygen), and incubated on an orbital shaker at 4 °C for 20 minutes. The homogenates were centrifuged at 14,000 rpm for 20 minutes at 4 °C, and the supernatants (protein lysates) were collected. For tissue culture cells, cells were harvested at sub-confluency and lysed with RIPA lysis buffer as described above. Protein concentrations were determined using the Bradford Protein Assay (Bio-Rad). Equivalent amounts of protein were resolved on precast Mini-PROTEAN TGX 7.5% gels (Bio-Rad) and transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST). The primary antibodies that were used are summarized in Table S1. Horseradish peroxidase-conjugated secondary antibodies (1:10,000, Jackson ImmunoResearch) and chemiluminescent substrates (Clarity ECL Substrates; Bio-Rad) were used for visualization on a Bio-Rad ChemiDoc Imaging System.

Cells were lysed with a lysis buffer (1% Brij58, 150 mM NaCl, 2 mM CaCl₂, 2 mM MgCl₂, 20 mM HEPES pH 7.4) and protein concentration was measured using BCA method (Thermo Fisher Scientific). Frozen skin was cut into small pieces on ice using razor blades, homogenized in 300 μL RIPA buffer (25mM Tris [pH 8], 150mM NaCl, 0.1%SDS, 0.5% sodium deoxycholate, 1%Triton X-100) with protease and phosphatase inhibitors using a plastic pestle (Axygen), and incubated on an orbital shaker at 4°C for 20 minutes. Homogenates were centrifuged at 14,000 rpm for 20 minutes at 4°C, and supernatants (protein lysates) collected. Protein concentrations were determined using the Bradford Protein Assay (Bio-Rad). Equivalent amounts of protein were resolved on precast Mini-PROTEAN TGX 7.5% gels (Bio-Rad) and transferred to PVDF membranes (Millipore). Membranes were blocked with 5% nonfat dry milk in TBST (Tris-buffered saline with 0.1% Tween-20). Primary antibodies used were summarized in Supplementary Table 1. Horseradish peroxidase-conjugated secondary antibodies (1:10,000) (Jackson ImmunoResearch) and chemiluminescent substrates (Clarity ECL Substrates; Bio-Rad) were used to visualize on a Bio-Rad ChemiDoc Imaging System.